|Budget Amount *help
¥2,900,000 (Direct Cost : ¥2,900,000)
Fiscal Year 1998 : ¥1,300,000 (Direct Cost : ¥1,300,000)
Fiscal Year 1997 : ¥1,600,000 (Direct Cost : ¥1,600,000)
We previously cloned and characterized the bovine lysozyme 5A (lys 5A) promoter with the purpose of determining cis-and trans-acting elements controlling airway epithelial cell-specific expression. We found that such expression is controlled by protein binding to an ets consensus sequence located-at-46/-40 from the transcription start site. The identity of the ets-related protein responsible for gene transactivation was unknown. In the present study, we screened ets-related proteins : MEF, ESE-1, Elf-1, Ets-1, Ets-2, and PEA 3 by transient transfection into epithelial cells and fibroblasts. Results showed that among these factors, MEF most strongly stimulated transcription in lung epithelial cells (A549), colon carcinoma cells (Caco2), and skin fibrobalsts (NIH3T3). Gel shift analysis of epithelial cell nuclear extracts using a lys 5A probe including the ets binding site (-50/-31) yielded a single band with retarded mobility. This band was supershifted by an antibody directed against MEF.This indicated that MEF is present in lung epithelial cells A549 and binds to the ets binding site in the lys 5A promoter. Supporting this, we found that anti-sense MEF mRNA decreased lys 5A promoter activity. Moreover, MEF overexpression in stable transfectants increased lysozyme mRNA and protein expression. In summary, these studies provide the first information identifying a transcription factor controlling lysozyme expression in epithelial cells. As bacteria become progressively more resistant to exogenously applied antibiotics, information regarding mechanisms controlling innate immunity, such as that provided by epithelial lysozyme, may offer important new therapeutic approaches.